The tumor antigen (T antigen) encoded by the viral A gene of the small oncogenic virus, SV40, is a large molecular weight polypeptide which is expressed in cells lytically infected with SV40 and in cells stably transformed by SV40. T antigen is thought to be responsible for regulating both viral DNA replication and transcription as well as for initiating and perhaps maintaining the transformed phenotype in a variety of cells. The aim of this research is to understand how T antigen carries out these apparently diverse and biologically significant functions. A battery of biochemical techniques in combination with recently developed cloning methods will be used to study the mechanism by wich T-antigen regulates the initiation of viral DNA replication and transcription. In addition, the influence of T antigen on cellular DNA replication will be investigated as a first step toward understanding the process of virally induced cellular transformation. First, I will compare the structure and specific DNA binding properties of T antigen proteins isolated in pure form from a variety of sources such as cells lytically infected with SV40, cells transformed by SV40 and cells infected with Adenovirus-SV40 hybrids. Details of the interactions between T antigen and DNA will be determined by identifying SV40 and cellular sequences which are specifically protected by T antigen from nuclease digestion and dimethylsulfate methylation. Next, other biological activities associated with the SV40 T-antigen will be probed by techniques such as micro-injection of macromolecules into individual cells in culture and isolation of in vitro nuclear systems which continue to support viral replication and transcription. Finally, the ability of purified T antigen to immunize animals against SV40 induced tumors (TSTA activity) will be investigated.